03 Astari Preparasi Sampel Biologi
BIOLOGICAL SAMPLE
PREPARATION
FOR TEM OBSERVATION
TEM Seminar
Nov 16, 2017
Astari Dwiranti, Ph.D
Why do we need EM for biological samples?
(O'Connor and Adams, 2010)
Why do we need EM for biological samples?
High
Resolution
High
magnification
High Depthof-field
Inner structure
investigation
3D
construction
light
electron
Seeing with electrons!
Biological samples types
• Animal tissues (ex. Liver, spleen, bone, etc)
• Cell culture
• Bacteria, biofilms
• Cellular organelles
• Plant tissues (ex. Seeds, pollen, leaves, etc).
SEM vs TEM
SEM
TEM
Imaging
Beam formation
Direct imaging
Accelerating voltage
50-30,000 volts
~120,000 volts
Specimen
Need not be thin
thin
Image
3D
2D
Magnification
~2 million
~50 million
Resolution
~ 0.4 nm
~ 0.5 Å
Biological samples
• Non-electronically
conductive
• Contains water
• Soft and can easily be
destroyed
• Various size of
specimen.
Goals of sample preparation
Observe sample near natural state as possible
Preservation of as many features as possible
Avoid artifacts (change, loss, or additional information)
Render specimen stable for examination in EM
Conventional TEM sample preparation
Fixation
•1st: aldehyde
•2nd: OsO4
•3rd: Tannic acid
1
Resin infiltration
Epoxy or acrylic
2
Dehydration
EtOH series
3
Sectioning
4
Embedding
5
6
Post-staining
Lead citrate
Uranyl acetate
Sample preparation for TEM
http://www.research.utah.edu/advanced-microscopy/education/electron-micro/index.html
Fixation
• preserve the sample structure in a state that most closely
resembles the structure of the original living state
(minimum alteration)
• protect and stabilize sample structure from changes
during subsequent treatments and irradiation.
Characteristics of a good fixative:
• Permeates cells readily and acts quickly
• Is irreversible
• Does not cause artifacts
Methods of fixation:
• Chemical fixation
• Freeze-fixation
Fixation
Glutaral
dehyde
PFA
Standard TEM fix: 2.5% GA + (2~4) % PFA,
30 min ~ overnight
Irreversible crosslinking of proteins
Reversible cross-linking,
small molecule,
penetrates quicker
Consideration of chemical fixation
• pH (isoelectric point)
• Total ionic strength of reagents
• Osmolarity
• Temperature
• Length of fixation
• Method of application of fixative.
Artefacts
Over-fixing
Muscle tissues fixed for 3 days at RT in 2.5% glutaraldehyde
(Knott, 2009)
Freeze fixation
Reasons:
• Halt rapid events
• Structures are
fixative sensitive
• Removal of water
changes morphology.
Disadvantages:
• Specialized equipment
required
• Freeze damage
• Limited view of specimen
• Hazards.
(Knott, 2009)
Secondary fixation
• Osmium tetroxide is a
heavy metal that fixed
unsaturated lipids.
• Used as both a
secondary fixative and
an electron stain
• Significantly improves
specimen preservation
and contrast.
Dehydration
• The process of gradually
replacing water in the sample
with a solvent (acetone or
ethanol).
• The solvent is then gradually
replaced with resin.
35 % EtOH
50 % EtOH
70 % EtOH
95 % EtOH
100 % EtOH
Propylene Oxide
1:1 Propylene Oxide : Epon resin
The dehydrating
times should be
adjusted to size
and kind of tissue.
Embedding
• Epoxy resin – araldite
• Acrylic resin – methyl
methycrylate.
Embedding with Epon (1-3 hours)
The ‘block’ are cured for 48 hours in a 60 degree oven.
Sectioning
Block trimming with a glass knife
• Sample is cut with ultramicrotome
• Thin sections (70nm)
• Place on grids.
Post staining
• Increases the image contrast
• Lead citrate (stains
polysaccharides, eg. glycogen)
• Uranyl acetate (basic stain eg
DNA).
No post-staining
Post-staining
(Oguro and Ghazizadeh, 2015)
Staining enhances the image contrast
Very small samples
do not need
embedding/sectioning
• Coat grids with plastic film
and carbon.
• Apply the particulate
specimens (eg. Protein,
viruses, DNA).
• Stain with heavy metal soln.
(eg. Uranyl acetate).
(Equilibrina, et al. 2015)
The procedure for every biological
sample may be different
(need to be adjusted
to obtain the best results).
Other
methods:
Hexamethyldisilazane (HMDS)
Ionic Liquids
Protein localization (Immunogold labelling)
• Secondary antibody is conjugated
to a colloidal gold particle.
• For cells and tissue, post-
embedding labelling is usually the
best option.
• The osmium tetroxide step is
omitted.
• Acrylic resins are used instead of
epoxy resin.
3D Tomography
Multi-lysosomal body
A.J. Koster and W.J.C. Geerts, Utrecht University
STEM tomography
Schematic diagram of STEM
Single axis tilt series
Back projection
STEM tomography would provide a 3D data of
the samples in a rapid and practical manner.
27
28
Tomography images of chromosomes
(Dwiranti, 2016)
Conclusions
• The general procedure for biological sample
preparation are fixation, staining, dehydration,
and sectioning.
• Different sample may require different
preparation method.
• Good preparation considering the character
of the samples would give the best TEM
images.
Life Science Facilities
-Ultramicrotome
-Chemical Kit
--> Epoxy Resin
Effects of aldehydes fixatives
Glutaraldehyde
Formaldehyde
TEM images of rat liver
(Knott, 2009)
IL method
Chromosomes
Metal coated
Uncoated
Artifact
Charged-up
Ionic liquid method
y = 0.7007x + 11.184
r2= 0.98093***
(Dwiranti, 2012)
36
The effect of OsO4 coating
Chromatin diameter
dynamics for different
OsO4 coating times
observed bySEM. Graph
shows the effect of OsO4
coating time on mean
chromatin diameter.
(Dwiranti, 2013)
37
Osmium tetroxide
• Non-polar tetrahedral molecule MW 254, solubility in water
and a variety of organic compounds.
• Ability to stabilize and stain lipids
• Mode of action: reacts primarily with double bonds and
sulfihydryl groups of proteins, causing major conformational
changes in the structure of proteins.
Chromosome 3D visualization by STEM tomography
IL method
Fixation
IL treatment
SEM
Time requirement: 50 min
IL method is applicable for
chromosome 3D observation
by STEM tomography.
Bar: 1 μm
39
Artifacts induced during TEM
preparation and observation
40
PREPARATION
FOR TEM OBSERVATION
TEM Seminar
Nov 16, 2017
Astari Dwiranti, Ph.D
Why do we need EM for biological samples?
(O'Connor and Adams, 2010)
Why do we need EM for biological samples?
High
Resolution
High
magnification
High Depthof-field
Inner structure
investigation
3D
construction
light
electron
Seeing with electrons!
Biological samples types
• Animal tissues (ex. Liver, spleen, bone, etc)
• Cell culture
• Bacteria, biofilms
• Cellular organelles
• Plant tissues (ex. Seeds, pollen, leaves, etc).
SEM vs TEM
SEM
TEM
Imaging
Beam formation
Direct imaging
Accelerating voltage
50-30,000 volts
~120,000 volts
Specimen
Need not be thin
thin
Image
3D
2D
Magnification
~2 million
~50 million
Resolution
~ 0.4 nm
~ 0.5 Å
Biological samples
• Non-electronically
conductive
• Contains water
• Soft and can easily be
destroyed
• Various size of
specimen.
Goals of sample preparation
Observe sample near natural state as possible
Preservation of as many features as possible
Avoid artifacts (change, loss, or additional information)
Render specimen stable for examination in EM
Conventional TEM sample preparation
Fixation
•1st: aldehyde
•2nd: OsO4
•3rd: Tannic acid
1
Resin infiltration
Epoxy or acrylic
2
Dehydration
EtOH series
3
Sectioning
4
Embedding
5
6
Post-staining
Lead citrate
Uranyl acetate
Sample preparation for TEM
http://www.research.utah.edu/advanced-microscopy/education/electron-micro/index.html
Fixation
• preserve the sample structure in a state that most closely
resembles the structure of the original living state
(minimum alteration)
• protect and stabilize sample structure from changes
during subsequent treatments and irradiation.
Characteristics of a good fixative:
• Permeates cells readily and acts quickly
• Is irreversible
• Does not cause artifacts
Methods of fixation:
• Chemical fixation
• Freeze-fixation
Fixation
Glutaral
dehyde
PFA
Standard TEM fix: 2.5% GA + (2~4) % PFA,
30 min ~ overnight
Irreversible crosslinking of proteins
Reversible cross-linking,
small molecule,
penetrates quicker
Consideration of chemical fixation
• pH (isoelectric point)
• Total ionic strength of reagents
• Osmolarity
• Temperature
• Length of fixation
• Method of application of fixative.
Artefacts
Over-fixing
Muscle tissues fixed for 3 days at RT in 2.5% glutaraldehyde
(Knott, 2009)
Freeze fixation
Reasons:
• Halt rapid events
• Structures are
fixative sensitive
• Removal of water
changes morphology.
Disadvantages:
• Specialized equipment
required
• Freeze damage
• Limited view of specimen
• Hazards.
(Knott, 2009)
Secondary fixation
• Osmium tetroxide is a
heavy metal that fixed
unsaturated lipids.
• Used as both a
secondary fixative and
an electron stain
• Significantly improves
specimen preservation
and contrast.
Dehydration
• The process of gradually
replacing water in the sample
with a solvent (acetone or
ethanol).
• The solvent is then gradually
replaced with resin.
35 % EtOH
50 % EtOH
70 % EtOH
95 % EtOH
100 % EtOH
Propylene Oxide
1:1 Propylene Oxide : Epon resin
The dehydrating
times should be
adjusted to size
and kind of tissue.
Embedding
• Epoxy resin – araldite
• Acrylic resin – methyl
methycrylate.
Embedding with Epon (1-3 hours)
The ‘block’ are cured for 48 hours in a 60 degree oven.
Sectioning
Block trimming with a glass knife
• Sample is cut with ultramicrotome
• Thin sections (70nm)
• Place on grids.
Post staining
• Increases the image contrast
• Lead citrate (stains
polysaccharides, eg. glycogen)
• Uranyl acetate (basic stain eg
DNA).
No post-staining
Post-staining
(Oguro and Ghazizadeh, 2015)
Staining enhances the image contrast
Very small samples
do not need
embedding/sectioning
• Coat grids with plastic film
and carbon.
• Apply the particulate
specimens (eg. Protein,
viruses, DNA).
• Stain with heavy metal soln.
(eg. Uranyl acetate).
(Equilibrina, et al. 2015)
The procedure for every biological
sample may be different
(need to be adjusted
to obtain the best results).
Other
methods:
Hexamethyldisilazane (HMDS)
Ionic Liquids
Protein localization (Immunogold labelling)
• Secondary antibody is conjugated
to a colloidal gold particle.
• For cells and tissue, post-
embedding labelling is usually the
best option.
• The osmium tetroxide step is
omitted.
• Acrylic resins are used instead of
epoxy resin.
3D Tomography
Multi-lysosomal body
A.J. Koster and W.J.C. Geerts, Utrecht University
STEM tomography
Schematic diagram of STEM
Single axis tilt series
Back projection
STEM tomography would provide a 3D data of
the samples in a rapid and practical manner.
27
28
Tomography images of chromosomes
(Dwiranti, 2016)
Conclusions
• The general procedure for biological sample
preparation are fixation, staining, dehydration,
and sectioning.
• Different sample may require different
preparation method.
• Good preparation considering the character
of the samples would give the best TEM
images.
Life Science Facilities
-Ultramicrotome
-Chemical Kit
--> Epoxy Resin
Effects of aldehydes fixatives
Glutaraldehyde
Formaldehyde
TEM images of rat liver
(Knott, 2009)
IL method
Chromosomes
Metal coated
Uncoated
Artifact
Charged-up
Ionic liquid method
y = 0.7007x + 11.184
r2= 0.98093***
(Dwiranti, 2012)
36
The effect of OsO4 coating
Chromatin diameter
dynamics for different
OsO4 coating times
observed bySEM. Graph
shows the effect of OsO4
coating time on mean
chromatin diameter.
(Dwiranti, 2013)
37
Osmium tetroxide
• Non-polar tetrahedral molecule MW 254, solubility in water
and a variety of organic compounds.
• Ability to stabilize and stain lipids
• Mode of action: reacts primarily with double bonds and
sulfihydryl groups of proteins, causing major conformational
changes in the structure of proteins.
Chromosome 3D visualization by STEM tomography
IL method
Fixation
IL treatment
SEM
Time requirement: 50 min
IL method is applicable for
chromosome 3D observation
by STEM tomography.
Bar: 1 μm
39
Artifacts induced during TEM
preparation and observation
40